Withanolides useful for the treatment of neurodegenerative diseases

ABSTRACT

Provided herein are synthetic analogs of withanolide natural products of formula (I), wherein R1-R4 are as defined herein, and their pharmaceutical uses in treating neurodegenerative diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/908,455, filed Nov. 25, 2013,which application is incorporated herein by reference in its entirety.

BACKGROUND

Technical Field

This application relates to synthetic analogs of withanolide naturalproducts and their pharmaceutical uses.

Description of the Related Art

Neurodegenerative diseases are characterized by selectiveneurodegeneration in specific regions of the brain and spinal cord.Amyotrophic Lateral Sclerosis (ALS), commonly known as “Lou Gehrig'sdisease”, is a progressive neurodegenerative disease of unknownetiology. The disease progressively impairs an individual's ability tocontrol voluntary muscle movement. The disease tends to progressrapidly, leading to paralysis and death within 2-5 years of diagnosis inmost cases. There are currently few therapeutic options for patientssuffering from ALS. The only FDA approved drug for the treatment of ALSis Rilutek®, introduced in 1995, which extends life expectancy inindividuals with ALS for a few months.

A number of hypotheses have been advanced concerning the pathogenesis ofALS. One is that glutamate, the most abundant excitatoryneurotransmitter in the central nervous system (CNS), causes neuronalcell death when its levels are chronically elevated. Glutamate levelshave been shown to be elevated in ALS patients (A. Platitakis and J. T.Caroscio, Ann. Neurol. 1987, 22: 5575-579). Oxidative stress is anotherarea of focus in ALS research. The potential importance of antioxidantdysfunction was triggered by the discovery that superoxide dismutase(SOD)1 mutations are associated with the familial form of ALS (D. R.Rosen et al., 1993, Nature, 362: 59-62) which account for about 20% ofcases. An autoimmune mechanism is another potential ALS pathogenesis (M.R. Pagani et al. Neurol. Res. Int., 2011, 2011:497080). Abnormal proteinmis-folding and aggregation have recently gained recognition as anunderlying pathogenic mechanism in ALS and several otherneurodegenerative diseases. Intracellular proteins that exhibitconformational mis-folding in ALS include SOD1 and transactive response(TAR) DNA-binding protein-43 (TDP-43).

Several years ago it was demonstrated that abnormalities in TDP-43, ahighly-conserved nuclear protein, were closely associated with ALS (T.Arai et al, 2006, Biochem. Biophys. Res. Commun., 351: 602-611). Inhealthy nerve cells, TDP-43 intracellular distribution is restricted tothe nuclear region. However, in ALS-affected neuronal cells, TDP-43 wasalso prominently present within cytoplasmic aggregates. Further, it wasshown that neuropathology-associated TDP-43 was atypicallyphosphorylated, extensively ubiquitinated, and proteolytically cleavedto generate carboxyl-terminus fragments in affected brain regions (M.Neumann et al., 2006, Science 314:130-133). Thus, the modified TDP-43accumulation patterns as well as intracellular processing abnormalitieswere proposed as contributors to degenerative neuronal cell changes inALS. These and other abnormalities related to TDP-43 are referred toherein as TDP-43 proteinopathies.

A relatively recent discovery related to TDP-43 has provided fundamentalinsights into pathogenic mechanisms operative in ALS. Studies performedat Laval University showed that TDP-43 was unexpectedly associated withthe p65 sub-unit of the nuclear factor-κB (NF-κB)inflammation-regulating transcription factor in spinal cord samplesobtained from ALS patients (V. Swamp et al., 2011, J. Exp. Med.,208:2429-2447).

Activation of the NF-κB signalling pathway is triggered by a number ofstimuli including reactive oxygen species, various pro-inflammatorycytokines including interleukin-1 (IL-1) and tumor necrosis factorα(TNFα) as well as different bacterial products (S. Vallabhapurapu and M.Karin, 2009, Annu. Rev. Immunol., 27: 693-733; L. Verstrepen et al.,2008, Cell. Mol. Life Sci. 65: 2964-29678). NF-κB activity is primarilyrestrained by its physical interaction with inhibitory IκB proteins. Inresting cells, NF-κB is present as a latent, inactive, IκB-bound complexin the cytoplasm. When a cell receives a threshold level of one of thesesignals, NF-κB is rapidly liberated from IκB, enters the nucleus andactivates transcription of specific genes, many of which encodepro-inflammatory and immune-response regulatory proteins. Almost allsignals that trigger the NF-κB signalling pathway converge on activationof a molecular complex that contains a serine residue-specific IκBkinase (IKK) (M. Adli et al., 2010, PLoS One, 5: e9428). In theclassical NF-κB pathway, activation of the IKK complex leads tophosphorylation mediated by IKKβ of two specific serines near the Nterminus of IκBα, which subsequently targets IκBα for intracellularubiquitination and degradation by the 26S proteasome complex(Vallabhapurapu 2009 op cit.; M. Adli 2010 op cit.). Activation of theNF-κB signalling pathway is generally a transient cellular event andtightly regulated (Vallabhapurapu 2009 op cit).

TDP-43 and the p65 chain of NF-κB were shown to haveco-immunoprecipitated in cell culture systems, spinal cord extracts fromtransgenic TDP-43 mice and spinal cord samples prepared from post-mortemALS patients, but not from matched control samples (Swamp 2011 op.cit.). In mouse and human spinal cord samples, p65 tended to co-localizewith TDP-43 in the nuclei of microglia, astrocytes and neurons. TDP-43mRNA levels were up-regulated by 2.5-fold while NF-κB mRNA wasup-regulated by approximately four-fold in ALS spinal cord samples ascompared to control subject material (Swamp 2011 op. cit.). Gel-shiftassays confirmed that the p65 chain of NF-κB p65 was more likely to bindto its consensus sequence of reporter DNA in the presence of TDP-43.Further, TDP-43 over-expression boosted production of pro-inflammatorycytokines, which heightened neuronal susceptibility to neurotoxicelements. Deletion mutation protein-mapping studies revealed that TDP-43interacted with the p65 chain component of NF-κB through its N-terminaldomain and RNA recognition motif (RMM-1) (Swamp 2011 op. cit.). NF-κBinhibition attenuated the vulnerability of cultured neuronsover-expressing TDP-43 to glutamate-induced or microglia cell-mediatedtoxicity.

Pharmacological intervention with withaferin A (WA) attenuated diseasesymptoms and ameliorated motor dysfunction in TDP-43 transgenic mice. WAwas shown to inhibit TNFα induced activation of IκB kinase β (IKKβ) viaa thioalkylation-sensitive redox mechanism (W. Vanden Berghe et al.,2012, Biochem. Pharmacol., 84: 1282-12891). IKKβ Ser-181hyperphosphorylation induced by WA led to inhibition of IκBαphosphorylation and degradation which prevented NF-κB translocation,NF-κB/DNA binding and gene transcription (M. Kaileh et al., 2007, J.Biol. Chem., 282: 4253-4264).

Withaferin A (WA) was the first withanolide-type compound isolated fromleaves of the Withania somifera plant.

This compound has been noted for its anti-inflammatory, anti-tumor,anti-angiogenic and immuno-suppressive activities. WA is a member ofwithanolides, which are generally described as a group of naturallyoccurring C28-steroidal lactone triterpenoids built on an intact orrearranged ergostane framework, in which C-22 and C-26 are appropriatelyoxidized to form a six-membered lactone ring (M. H. Mirjalili et al.,Molecules 2009, 14(7): 2373-2393). Numerous analogs of WA have beenpurified from withanolide-containing plant material, synthesized, orsemi-synthetically prepared from the WA starting material (U.S. Pub. No.2011/0230551).

WA has been proposed as a treatment for neurodegenerative diseases, suchas ALS, frontotemporal lobar degeneration, Parkinson's disease andAlzheimer's disease (WO2012/174666) and it has been shown that WA iseffective in ameliorating disease progression in mouse models of ALS. Anin vivo therapeutic effect of WA through NF-κB inhibition has beendemonstrated in four recognized transgenic mouse models of ALS.

Although WA is a promising therapeutic agent for the treatment of ALSand other neurodegenerative diseases, it has a short half life whenadministered in vivo, as well as some toxicity. Hence there is a needfor novel compounds with improved pharmacokinetic, bio-distribution andsafety profiles.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows bioluminescense imaging of the brains of GFAP-luciferasetransgenic mice after exposure to lipopolysaccharide (LPS) followed bytreatment with saline or the withanolide compounds 4-O-methyl WA or27-O-methyl WA.

FIG. 1B is a bar graph showing the relative intensity of thebioluminescense of the brain images from FIG. 1A.

FIG. 2 includes bar graphs showing the effect of different concentrationof withanolides 4-O-methyl WA, 27-O-methyl WA and 4,27-O-dimethyl A onNF-κB reporter activity in BV2 microglial cells stimulated with LPS.

FIG. 3 includes bar graphs showing the effect of differentconcentrations of withanolides 4-O-methyl WA, 27-O-methyl WA and4,27-O-dimethyl WA on the up-regulation of TNF-α-induced signallingactivity in the HEK²⁹³-NF-κB-luciferase reporter cell line.

FIG. 4A shows the accelerating rotarod performance of TDP-43 A315T micetreated with vehicle, 4-O-Methyl WA or 27-O-Methyl WA analogs over a 15week period.

FIG. 4B shows the linear regression analysis of the data in FIG. 4A.

SUMMARY

Described herein are semi-synthetic analogs of withaferin A and methodsfor using the analogs for the prophylaxsis or treatment ofneurodegenerative diseases or conditions such as Amyotrophic LateralSclerosis (ALS), frontotemporal lobar degeneration (FTLD), Parkinson'sdisease, Alzheimer's disease and mild cognitive impairment.

Accordingly, in one aspect, the invention is directed to compounds ofFormula (I):

wherein:

-   -   R¹ is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        heterocyclylalkyl, —R^(a)—OR^(b), —C(O)R^(b), cycloalkylalkyl or        —P(O)₂O²⁻;    -   R² is hydrogen, alkyl, alkenyl, haloalkyl, —OR^(b) or        —OC(O)R^(b);    -   R³ is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        heterocyclylalkyl, —R^(a)—OR^(b), —C(O)R^(b), cycloalkylalkyl or        —P(O)₂O²⁻;    -   R⁴ is hydrogen, alkyl, alkenyl, haloalkyl, —OR^(b) or        —OC(O)R^(b);    -   R^(a) is an alkylene or alkenylene chain; and    -   R^(b) is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        cycloalkylalkyl or heterocyclylalkyl,    -   as an isolated stereoisomer or mixture thereof, or a        pharmaceutically acceptable salt thereof, provided that,    -   when R² and R⁴ are each hydrogen, R¹ and R³ cannot both be        selected from the group consisting of hydrogen and —C(O)CH₃; or    -   when R¹ and R³ are each —C(O)CH₃, R² or R⁴ cannot both be        selected from the group consisting of hydrogen and —OC(O)CH₃.

In some embodiments, the compounds are of formula (I) wherein R² and R⁴are each hydrogens, and the compounds have a structure represented byFormula (Ia):

wherein:

-   -   R¹ is hydrogen, alkyl or alkenyl; and    -   R³ is hydrogen, alkyl or alkenyl.

In further embodiments, the compounds are: 27-O-methylwithaferin A (R¹is hydrogen and R³ is methyl), 4-O-methylwithaferin A (R¹ is methyl andR³ is hydrogen), and 4,27-O-dimethylwithaferin A (R¹ is methyl and R³ ismethyl).

Another aspect of the invention is directed to a pharmaceuticalcomposition comprising a compound of formula (I) or formula (Ia) and apharmaceutically acceptable excipient.

In another aspect, the invention is directed to a method of treating orpreventing a disease characterized by TDP-43 proteinopathy in a patientcomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of a compound of formula (I) orformula (Ia).

In another aspect, the invention is directed to a method of treating orpreventing amyotrophic lateral sclerosis in a patient comprisingadministering to a patient in need thereof a therapeutically orprophylactically effective amount of a compound of formula (I) orformula (Ia).

In a further aspect, the invention is directed to a method of treatingor preventing Alzheimer's disease in a patient comprising administeringto a patient in need thereof a therapeutically or prophylacticallyeffective amount of a compound of formula (I) or formula (Ia).

In a further aspect, the invention is directed to a method of treatingor preventing Parkinson's disease in a patient comprising administeringto a patient in need thereof a therapeutically or prophylacticallyeffective amount of a compound of a formula (I) or formula (Ia).

In another aspect, the invention is directed to method of treating orpreventing motor neuron disease in a patient comprising administering toa patient in need thereof a therapeutically or prophylacticallyeffective amount of a compound of formula (I) or formula (Ia).

In another aspect, the invention is directed to a method of treating orpreventing frontotemporal lobar degeneration in a patient comprisingadministering to a patient in need thereof a therapeutically orprophylactically effective amount of a compound of formula (I) orformula (Ia).

In another aspect, the invention is directed to a method of treating orpreventing mild cognitive impairment or preventing the development ofAlzheimer's disease in a patient exhibiting mild cognitive impairmentcomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of a compound of Formula (I) orFormula (Ia).

DETAILED DESCRIPTION

Disclosed herein are semi-synthetic analogs of Withaferin A and theirvarious pharmaceutical uses, particularly in treating neurodegenerativediseases, including amyotrophic Lateral Sclerosis (ALS), frontotemporallobar degeneration (FTLD), Parkinson's disease, mild cognitiveimpairment (MCI), Alzheimer's disease, and diseases associated withTPD-43 proteinopathy. One embodiment provides a compound of Formula (I):

wherein:

-   -   R¹ is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        heterocyclylalkyl, —R^(a)—OR^(b), —C(O)R^(b), cycloalkylalkyl or        —P(O)₂O²⁻;    -   R² is hydrogen, alkyl, alkenyl, haloalkyl, —OR^(b) or        —OC(O)R^(b);    -   R³ is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        heterocyclylalkyl, —R^(a)—OR^(b), —C(O)R^(b), cycloalkylalkyl or        —P(O)₂O²⁻;    -   R⁴ is hydrogen, alkyl, alkenyl, haloalkyl, —OR^(b) or        —OC(O)R^(b);    -   R^(a) is an alkylene or alkenylene chain; and    -   R^(b) is hydrogen, alkyl, alkenyl, haloalkyl, aralkyl,        cycloalkylalkyl or heterocyclylalkyl,    -   as an isolated stereoisomer or mixture thereof, or a        pharmaceutically acceptable salt thereof, provided that,    -   when R² and R⁴ are each hydrogen, R¹ and R³ cannot both be        selected from the group consisting of hydrogen and —C(O)CH₃; or    -   when R¹ and R³ are each —C(O)CH₃, R² or R⁴ cannot both be        selected from the group consisting of hydrogen and —OC(O)CH₃.

In various embodiments, the compound of Formula (I) comprises one ormore alkyl ether moieties, in particular, at the C-4, C-27 or bothlocations.

In certain embodiments, Wand R³ are independently lower alkyl oralkenyl. In further embodiments, R¹ and R³ are independently methyl.

In various embodiments, R² and R⁴ are each hydrogens, and the compoundof Formula (I) has a structure represented by Formula (Ia):

wherein:

-   -   R¹ is hydrogen, alkyl or alkenyl; and    -   R³ is hydrogen, alkyl or alkenyl.

In further embodiments, the compounds are: 27-O-methylwithaferin A(R¹ ishydrogen and R³ is methyl), 4-O-methylwithaferin A (R¹ is methyl and R³is hydrogen), and 4,27-O-dimethylwithaferin A (R¹ is methyl and R³ ismethyl)

Each individual compound disclosed in U.S. Pub. No. 2011/0230551 isexpressly excluded from the scope of Formulae (I) and (Ha).

Another embodiment provides a pharmaceutical composition comprising acompound of Formula (I) or Formula (Ia), as defined herein, and apharmaceutically acceptable excipient.

Various embodiments further provide pharmaceutical use of the compoundof Formula (I) or (Ia). More specifically, the pharmaceutical uses ofthe compound or composition comprising the same include the treatment ofneurodegenerative diseases, or prevention of the progression orworsening of neurodegenerative diseases. In particular, theneurodegenerative disease is characterized by TDP-43 proteinopathy.

Thus, one embodiment provides a method of treating or preventing adisease characterized by TDP-43 proteinopathy in a patient comprisingadministering to a patient in need thereof a therapeutically orprophylactically effective amount of a compound of Formula (I) orFormula (Ia).

A further embodiment provides a method of treating or preventingamyotrophic lateral sclerosis in a patient comprising administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of a compound of Formula (I) or Formula (Ia).

Another embodiment provides a method of treating or preventingAlzheimer's disease in a patient comprising administering to a patientin need thereof a therapeutically or prophylactically effective amountof a compound of Formula (I) or Formula (Ia).

Another embodiment provides a method of treating or preventingParkinson's disease in a patient comprising administering to a patientin need thereof a therapeutically or prophylactically effective amountof a compound of Formula (I) or Formula (Ia).

Another embodiment provides a method of treating or preventing motorneuron disease in a patient comprising administering to a patient inneed thereof a therapeutically or prophylactically effective amount of acompound of Formula (I) or Formula (Ia).

Another embodiment provides a method of treating or preventingfrontotemporal lobar degeneration in a patient comprising administeringto a patient in need thereof a therapeutically or prophylacticallyeffective amount of a compound of Formula (I) or Formula (Ia).

Another embodiment provides a method of treating or preventing mildcognitive impairment or preventing the development of Alzheimer'sdisease in a patient exhibiting mild cognitive impairment comprisingadministering to a patient in need thereof a therapeutically orprophylactically effective amount of a compound of Formula (I) orFormula (Ia).

Definitions

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated:

“Amino” refers to the NH₂ radical.

“Carboxy” refers to the —C(O)OH radical.

“Cyano” refers to the —CN radical.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O radical.

“Thioxo” refers to the ═S radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to twelve carbon atoms, preferably one toeight carbon atoms or one to six carbon atoms and which is attached tothe rest of the molecule by a single bond, for example, methyl, ethyl,n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.For purposes of this invention, the term “lower alkyl” refers to analkyl radical having one to four carbon atoms.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing at least onedouble bond, having from two to twelve carbon atoms, preferably one toeight carbon atoms and which is attached to the rest of the molecule bya single bond, for example, ethenyl, prop-1-enyl, but-1-enyl,pent-1-enyl, penta-1,4-dienyl, and the like.

“Alkylene chain” refers to a straight or branched divalent hydrocarbonchain linking the rest of the molecule to a radical group, consistingsolely of carbon and hydrogen, containing no unsaturation and havingfrom one to twelve carbon atoms, for example, methylene, ethylene,propylene, n-butylene, and the like. The alkylene chain is attached tothe rest of the molecule through a single bond and to the radical groupthrough a single bond. The points of attachment of the alkylene chain tothe rest of the molecule and to the radical group can be through onecarbon in the alkylene chain or through any two carbons within thechain.

“Alkenylene chain” refers to a straight or branched divalent hydrocarbonchain linking the rest of the molecule to a radical group, consistingsolely of carbon and hydrogen, containing at least one double bond andhaving from two to twelve carbon atoms, for example, ethenylene,propenylene, n-butenylene, and the like. The alkenylene chain isattached to the rest of the molecule through a double bond or a singlebond and to the radical group through a double bond or a single bond.The points of attachment of the alkenylene chain to the rest of themolecule and to the radical group can be through one carbon or any twocarbons within the chain.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 14 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic, ortricyclic system and which may include spiro ring systems. An arylradical is commonly, but not necessarily, attached to the parentmolecule via an aromatic ring of the aryl radical. Aryl radicalsinclude, but are not limited to, aryl radicals derived fromacenaphthylene, anthracene, azulene, benzene,6,7,8,9-tetrahydro-5H-benzo[7]annulene, fluorene, as-indacene,s-indacene, indane, indene, naphthalene, phenalene, and phenanthrene.

“Aralkyl” refers to a radical of the formula —R_(a)—R_(c), where R_(a)is an alkylene chain as defined above and R_(c) is one or more arylradicals as defined above. Examples of aralkyl include, withoutlimitation, benzyl, diphenylmethyl and the like.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,which includes fused, spiro or bridged ring systems, having from threeto fifteen carbon atoms, preferably having from three to ten carbonatoms, more preferably from five to seven carbons and which is saturatedor unsaturated and attached to the rest of the molecule by a singlebond. For purposes of this invention, a bridged ring system is a systemwherein two non-adjacent ring atoms thereof are connected through anatom or a group of atoms, wherein the atom or the group of atoms are thebridging element. Examples of cycloalkyl include, without limitation,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Polycyclic radicals include fused, spiro or bridgedcycloalkyl radicals, for example, C₁₀ radicals such as adamantanyl(bridged) and decalinyl (fused), and C₇ radicals such asbicyclo[3.2.0]heptanyl (fused), norbornanyl and norbornenyl (bridged),as well as substituted polycyclic radicals, for example, substituted C₇radicals such as 7,7-dimethylbicyclo[2.2.1]heptanyl (bridged), and thelike.

“Cycloalkylalkyl” refers to a radical of the formula —R_(a)R_(d) whereR_(a) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above.

“Halo” refers to fluoro, chloro, bromo or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, for example,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ringsystem radical which comprises one to twelve carbon atoms and from oneto six heteroatoms selected from the group consisting of nitrogen,oxygen and sulfur. Unless stated otherwise specifically in thespecification, the heterocyclyl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include spiro or bridgedring systems; and the nitrogen, carbon or sulfur atoms in theheterocyclyl radical may be optionally oxidized; the nitrogen atom maybe optionally quaternized; and the heterocyclyl radical may be partiallyor fully saturated. Examples of a bridged heterocyclyl include, but arenot limited to, azabicyclo[2.2.1]heptanyl, diazabicyclo[2.2.1]heptanyl,diazabicyclo[2.2.2]octanyl, diazabicyclo[3.2.1]octanyl,diazabicyclo[3.3.1]nonanyl, diazabicyclo[3.2.2]nonanyl andoxazabicyclo[2.2.1]heptanyl. A “bridged N-heterocyclyl” is a bridgedheterocyclyl containing at least one nitrogen, but which optionallycontains up to four additional heteroatoms selected from O, N and S. Forpurposes of this invention, a non-bridged ring system is a systemwherein no two non-adjacent ring atoms thereof are connected through anatom or a group of atoms. Examples of heterocyclyl radicals include, butare not limited to, dioxolanyl, 1,4-diazepanyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl,octahydro-1H-pyrrolo[3,2-c]pyridinyl,octahydro-1H-pyrrolo[2,3-c]pyridinyl,octahydro-1H-pyrrolo[2,3-b]pyridinyl,octahydro-1H-pyrrolo[3,4-b]pyridinyl, octahydropyrrolo[3,4-c]pyrrolyl,octahydro-1H-pyrido[1,2-a]pyrazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolidinyl, oxazolidinyl, 3,7-diazabicyclo[3.3.1]nonan-3-yl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, thienyl[1,3]dithianyl,trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, azetidinyl,octahydropyrrolo[3,4-c]pyrrolyl, octahydropyrrolo[3,4-b]pyrrolyl,decahydroprazino[1,2-a]azepinyl, azepanyl, azabicyclo[3.2.1]octyl, and2,7-diazaspiro[4.4]nonanyl.

“Heterocyclylalkyl” refers to a radical of the formula R_(a)R_(e) whereR_(a) is an alkylene chain as defined above and R_(e) is a heterocyclylradical as defined above, and when the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkylene chain at the nitrogen atom.

As used herein, unless specified otherwise, each one of alkyl, alkenyl,haloalkyl, aralkyl, heterocyclylalkyl, alkylene chain, alkenylene chain,cycloalkylalkyl moieties may be optionally substituted, whereby one ormore hydrogens are replaced by one or more of the followingsubstituents: amino, halo, cyano, nitro, oxo, thioxo, trialkylsilanyl(including trimethylsilanyl), —OR^(f), —OC(O)—R^(f), —N(R^(f))₂,—C(O)R^(f), —C(O)OR^(f), —C(O)N(R^(f))₂, —N(R^(f))C(O)OR^(f),—N(R^(f))C(O)R^(f), —N(R^(f))S(O)₂R^(f), —S(O)_(t)OR^(f) (where t is 1or 2), —S(O)_(p)R^(f) (where p is 0, 1 or 2), and —S(O)₂N(R^(f))₂ whereeach R^(f) is independently selected from the group consisting of alkyl,alkenyl, haloalkyl, aralkyl, heterocyclylalkyl, cycloalkylalkyl.

“Patient” means a mammal who has been diagnosed with a neurodegenerativedisease or who is genetically predisposed to such diseases.

“Mammal” means any vertebrate of the class Mammalia. Humans and domesticanimals, such as cats, dogs, swine, cattle, sheep, goats, horses,rabbits, and the like are a particular focus. Preferably, for purposesof this invention, the mammal is a primate (e.g., monkey, baboon,chimpanzee and human), and more preferably, the mammal is a human.

“Pharmaceutically acceptable excipient” includes without limitation anyadjuvant, carrier, excipient, glidant, sweetening agent, diluent,preservative, dye/colorant, flavor enhancer, surfactant, wetting agent,dispersing agent, suspending agent, stabilizer, isotonic agent, solvent,or emulsifier which has been approved by the United States Food and DrugAdministration as being acceptable for use in humans or domesticanimals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, benethamine,benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine,triethanolamine, tromethamine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

A “pharmaceutical composition” refers to a formulation of a compound offormula (I) or a formulation of a therapeutic agent described herein anda medium generally accepted in the art for the delivery of thebiologically active compound to mammals, for example, humans. Such amedium includes all pharmaceutically acceptable carriers, diluents orexcipients therefor.

“Neurodegenerative disease” refers to a progressive loss of structure orfunction of neurons, including death of neurons. Examples of theneurodegenerative diseases include, without limitation, Parkinson's,Alzheimer's, ALS, motor neuron disease and frontotemporal lobardegenration (FTLD). In particular, the neurodegenerative disease may becharacterized by TDP-43 proteinopathy. Neurodegenerative disease alsoincludes mild cognitive impairment (MCI) (described in S. Gautier etal., 2006, The Lancet 351: 1262-1270).

“Therapeutically effective amount” refers to that amount of thetherapeutic agent sufficient to delay, forestall or minimize theprogress of neurodegeneration, or to provide a therapeutic benefit inthe treatment or management of neurodegenerative diseases, including theamelioration of symptoms associated with neurodegenerative diseases.

“Prophylactically effective amount” refers to that amount of theprophylactic agent sufficient to result in preventing or forestallingneurodegenerative diseases, particularly in patients who may begenetically predisposed to such diseases. A prophylactically effectiveamount may refer to the amount of prophylactic agent sufficient toprevent the age-related or early on-set of neurodegenerative diseases.

As used herein, the terms “prevent”, preventing” and “prevention” referto the prevention of the spread or onset of neurodegenerative diseasesin a patient.

As used herein, the terms “treat”, “treating” and “treatment” refer todelay, forestall or minimize the neurodegenerative process, preferablyprior to neurodegenerative diseases such as ALS, Parkinson's,Alzheimer's, FTLD or mild cognitive impairment (MCI) could develop fromthe neurodegeneration. The terms also refer to management ofneurodegenerative diseases, including the amelioration of symptomsassociated with neurodegenerative diseases.

The compounds of formula (I) and (Ia), or their pharmaceuticallyacceptable salts, may contain one or more asymmetric centers and maythus give rise to enantiomers, diastereomers, and other stereoisomericforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids. The present invention ismeant to include all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, such as HPLC usinga chiral column. When the compounds described herein contain olefinicdouble bonds or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

Preparation of the Compound of Formula (I)

The following reaction scheme shows a semi-synthetic approach topreparing the compound of Formula (Ia) by using Withaferin A as astarting material. More specifically, Withaferin A (available fromSigma-Aldrich Canada) can be treated with one or more alkylating agentsto alkylate the —OH groups of Withaferin A.

The reaction may produce a mixture of mono-alkylated compounds at C-4 orC-27 locations, as well as di-alkylated WA at both C-4 and C-27locations. The alkylated compounds may be separated and isolated byknown methods in the art. Where R¹ and R³ are identical, a singlealkylation step can be carried out. Where R¹ and R³ are different, twoalkylation steps may be carried out. For example, a step-wise reactionusing alkylating agents R¹—X and R³—X (X being a leaving group) may becarried out to separately alkylate the hydroxy groups of Withaferin A.

Selective protection of the hydroxy groups at C-4 or C-27 of WithaferinA can direct the alkylation step to a particular location. For instance,the C-4 hydroxy group may be first protected such that the alkylationstep only takes place at the C-27 hydroxy group.

Functionalization at the C-12 and C-15 locations of a compound ofFormula (I) may be carried out according to the methods disclosed inU.S. Pub. No. 2011/0230551, which reference is incorporated herein byreference in its entirety.

It will be appreciated by those skilled in the art that in the processesdescribed below the functional groups of intermediate compounds may needto be protected by suitable protecting groups. Such functional groupsinclude hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino include benzyl,t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acids include alkyl, aryl or arylalkyl esters.

Protecting groups may be added or removed in accordance with standardtechniques, which are known to one of ordinary skill in the art and asdescribed herein. The use of protecting groups is described in detail inGreene, T. W. and P. G. M. Wuts, Greene's Protective Groups in OrganicSynthesis (1999), 3rd Ed., Wiley. As one of skill in the art wouldappreciate, the protecting group may also be a polymer resin such as,but not limited to, a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Dosage

The amount of the withanolide compounds required for use in treatmentwill vary not only with the particular compound selected but also withthe route of administration, the nature of the condition for whichtreatment is required and the age and condition of the patient and willbe ultimately determined by a physician. In general, the amount of acompound required for use in treatment will vary not only with theparticular compound selected but also with the route of administration,the nature of the condition for which treatment is required and the ageand condition of the patient and will be ultimately be determined by aphysician. Generally, a suitable dose will be for example in the rangeof 0.01 to 1000 mg/kg of body weight per day, or for example, in therange of 0.1 to 100 mg/kg/day, or, for example, in the range of 0.5 to50 mg/kg/day, or for example, in the range of 1 to 25 mg/kg/day. Dosesmay be administered at appropriate intervals, for example as one, two,three, four or more doses per day. In some cases doses may beadministered every day, every two to three days, every four to fivedays, or every five to seven days. Dosing may continue for days, weeks,months or years, as required.

EXAMPLES Example 1 Preparation of WA Methyl Ether Analogs from WA

Preparation

150 mg of Withaferin A (available from Sigma-Aldrich Canada) (WA 1) weretreated with sodium hydride in methyl iodide. The reaction produced amixture of both mono-methylated compounds and di-methylated WA. Thereaction mixture was filtered to remove excess sodium hydride and sodiumiodide. The filtrate was dried and the residue re-dissolved indichloromethane. The resulting solution was chromatographed on a silicagel column. Fractions containing the mono-methyl ethers (1 and 3) anddimethyl ether (4) were combined and the compounds separated by reversedphase chromatography.

Pure fractions of each compound were pooled, concentrated, extractedinto dichloromethane and dried. Approximately 5-10 mg of each compoundwas thus obtained, each as a white solid. Spectroscopic data of thesecompounds are provided in Table 1 below.

TABLE 1 m/z ∂ ppm (CDCl₃) Compound [M + H]⁺ H-4 H-27a H-27b OH-4 OH-274-OMe 27-OMe Withaferin A (1) 471 3.78 4.41 4.35 2.52 2.86 — — (dd) (dd)(dd) (d) (t) 27-O-methyl 485 3.77 4.29 4.17 2.50 — — 3.39 withaferin A(2) (dd) (d) (d) (d) (s) 4-O-methyl 485 3.27 4.41 4.35 — 2.86 3.45 —withaferin A (3) (d) (dd) (dd) (t) (s) 4,27-O-dimethyl 499 3.27 4.294.17 — — 3.46 3.39 withaferin A (4) (d) (d) (d) (s) (s)

Example 2 Brain Bioluminescence in GFAP-Luciferase Mice Exposed to LPS

Two of the novel withanolides were tested for their therapeutic activityin vivo using a transgenic mouse model. Transgenic GFAP-luciferase micegenerated in the laboratory of Dr. J. P. Julien were used to assess theability of the withanolides to inhibit astrogliosis associated withinflammation induced by lipopolysaccharide (LPS) exposure. In vivobioluminescence imaging was performed to asses the inflammatory responsein the cranial region. A decrease in bioluminescense signals in theevaluated region in comparison to the control (saline) indicated thatthe withanolides had crossed the blood brain barrier and subsequentlyinhibited gliosis.

Withanolides [4-O-methyl withaferin A (4-O-methyl WA) and 27-O-methylwithaferin A (27-O-methyl WA)] were diluted in 100% dimethyl sulfoxide(DMSO) at a final concentration of 2 mg/ml. GFAP-luc transgenic micewere used to test in vivo the efficacy of these analogs. In thesetransgenic mice, the firefly luciferase reporter gene is under thecontrol of a 12 kb DNA fragment of the glial fibrillary acidic protein(GFAP) promoter (Caliper Life Sciences). The luciferase reporter isinducible following LPS administration resulting in GFAP transcriptionalregulation. If the administration of test compounds following theinjection of LPS decreases LPS-induced astrogliosis in GFAP-luc mice,this is visualized as a reduced bioluminescent signal upon imaging.

Transgenic mice under anesthesia were induced to inhale either 5 ul of asolution of LPS (1 mg/ml in 0.9% saline) or a solution of 0.9% salinealone followed 2 h later by an intraperitoneal (i.p.) injection of 0.5ml of the test compounds (10% dilution in 0.9% saline) at a finalconcentration of 4 mg/kg. The following day, 2 h before imaging (24 hafter the administration of the LPS solution), the transgenic mice wereagain injected with the withanolides as previously. Twenty minutes priorto imaging, the mice received an i.p. injection of the luciferasesubstrate D-luciferin (150 mg/kg). D-luciferin was dissolved in 0.9%saline to a final concentration of 20 mg/ml. Mice were anesthetized andimaged using an IVIS 200 imaging system (CaliperLS-Xenogen).

The results showed a significant decrease in the bioluminescent signalin mice treated with either 4-O-methyl WA or 27-O-methyl WA compared tomice treated with saline-DMSO alone (FIG. 1A). FIG. 1B shows a summarygraph of the quantification of the luciferase activity expressed ascounts of total photon emission in the brain of the GFAP-transgenic miceshown in FIG. 1A. This experiment demonstrated a significant reductionin astrogliosis in GFAP-transgenic mice treated with either 4-O-methylWA or 27-O-methyl WA.

Example 3

Novel withanolides inhibit NF-κB reporter activity in BV2 microglialcells stimulated with LPS. The withanolides 4-O-methyl WA, 27-O-methylWA and 4,27-O-dimethyl WA were tested for their ability to inhibit NF-κBactivation. An NF-κB specific luciferase reporter system in BV-2microglial cells was first established. This cell line was generated bystable transfection of BV-2 cells with stable insertion of a luciferasereporter 4 kBwt luciferase plasmid and subsequent selection withhygomycin. LPS was used to stimulate NF-κB activity in these cells.Twenty-five thousand hygromycin B-resistant BV-2 cells were seeded perwell in 24-well dishes and allowed to adhere overnight. The nextmorning, the culture medium (DMEM+10% FBS) was removed and 1 ml of freshmedium (DMEM without FBS) added to each well. The stock solutions ofwithanolides (2 mg/ml in DMSO) were diluted in 1×PBS to variousconcentration (0.05 to 5 uM) and added to the wells. LPS was added onehour later at a final concentration of 100 ng/ml. Four hours later, theBV-2 cells were rinsed with 1×PBS prior to proceeding with theluciferase assay that was performed according to the manufacturer'sinstructions (Bright-Glo™ luciferase assay system, Promega, Wis.).

The results, provided in FIG. 2, showed that all of the withanolidestested had the ability to inhibit NF-κB expression. Table 2 below showsthe IC50 of certain compounds of Formula (Ia).

TABLE 2 Compounds IC₅₀ (μM) 4-O-methyl WA 0.97 27-O-methyl WA 0.714,27-O-dimethyl WA 0.80 WA 0.50

Example 4 Novel Withanolides Inhibit Up-Regulation of TNF-A-InducedSignalling Activity in the HEK293-NF-κB-Luciferase Reporter Cell Line

Ten thousand hygromycin B-resistant HEK-293 cells were seeded per wellin a 96-well plate (Corning, New-York) and allowed to adhere overnight.The next morning, the culture medium (DMEM+10% FBS) was removed and 100ml of fresh medium (DMEM without FBS) added to each well. The stocksolutions of withanolides 4-O-methyl WA, 27-O-methyl WA and4,27-O-dimethyl WA (2 mg/ml in DMSO) were diluted in 1×PBS to variousconcentration (0.05 to 5 uM) and added to the wells. Human recombinantTNF-alpha (R&D Systems, Minneapolis) was added one hour later at a finalconcentration of 40 ng/ml. Four hours later, the HEK-293 cells wererinsed with 1×PBS prior to the luciferase assay, which was performedaccording to the manufacturer's instructions (Bright-Glo™ luciferaseassay system, Promega, Wis.).

The results of the assay, shown in FIG. 3, confirmed that 4-O-methyl WA,27-O-methyl WA and 4,27-O-dimethyl WA inhibited NF-κB expression. TheIC₅₀s for 4-O-methyl WA, 27-O-methyl WA and 4,27-O-dimethyl WA are shownin Table 3.

TABLE 3 Compounds IC₅₀ (μM) 4-O-methyl WA 0.32 27-O-methyl WA 0.264,27-O-dimethyl WA 0.45 WA 0.34

Example 5 Safety Comparison of WA, 4-O-Methyl WA and 27-O-Methyl WA

An acute dose-escalation study was carried out to compare thetolerability in vivo of WA, 4-0 methyl WA and 27-0 methyl WA. NormalC57BL/6 female mice were injected intraperitoneally with WA, 4-O-methylWA or 27-O-methyl WA at doses ranging from 20 to 65 mg/kg (20, 25, 30,35, 45, 55 and 65 mg/kg). Animals were observed for signs of morbidity,mortality and clinical changes in behavior, breathing, heartbeat,hydration and other conditions (such as ascites, shock, severe diarrheaand hemorrhage) at 1 h, 6 h and 24 hours post-dosing. Body weight wasmeasured pre-dosing and at 24 h.

The withanolides 4-O-methyl WA and 27-O-methyl WA both showed a muchbetter safety profile than WA. The LD50 (lethal dose, 50%) was found tobe 55 mg/Kg for WA. The same LD50 value (54 mg/kg) for WA was previouslyreported (Patwardhan et al., Drug Discovery and Development, (2006),edited by M. S. Chorghade, Wiley). In contrast, no mortality wasobserved when mice were administered 55 mg/kg of either 4-O-methyl WA or27-O-methyl WA. At the highest dose tested in the study, 65 mg/kg, nomortality was observed for mice injected with either 4-O-methyl WA or27-O-methyl WA. However, the animals in these groups did exhibit someweakness and a decrease in activity post-dosing.

Example 6 Withaferin a Analogs Inhibit Neurological Disease Developmentin a TDP-43 A315T Transgenic Mouse Model of ALS

The impact of repeated dosing with 4-O-Methyl WA and 27-O-Methyl WA onthe development of neurological disease in the TDP-43 A315T transgenicmouse model of ALS was evaluated. The TDP-43 A315T mouse model for ALSis fully described in Swamp, V et al., Brain 134: 2610-2626 2011. TDP-43A315T mice begin to exhibit neurological deficits at about 9 months ofage, as measured by their performance in a number of behavioral andmotor function tests (Barnes maze test, accelerating rotarod test andpassive avoidance test).

Male and female TDP-43 A315T mice of approximately 9 months and 25-55 gmwere included in the study, and were randomly assigned to three groups,as follows:

Group 1 (n=6) received 4-O-Methyl WA at 5 mg/kg;

Group 2 (n=7) (the control group) received vehicle (2% polysorbate[Tween-80]/5% DMSO/93% saline); and

Group 3 (n=7) received 27-O-Methyl WA at 5 mg/kg.

All animals received i.p. injections of either vehicle or 5 mg/kg of thetest WA analogs every 2 days for 15 weeks. The injection volume at 12.5mL/kg was based on the body weight of each individual animal at thestart of the study.

All animals were observed at least once daily throughout the study forclinical signs, and were individually weighed weekly before the rotarodtest. There were no significant changes in body weight or clinical signsover the study period. After 15 weeks, the mice were sacrificed, and theexpression of the mutant TPD-43A315T protein in the spinal cord of eachmouse was confirmed by an antibody immunofluorescence or western blottest.

Accelerating rotarod (as described in Gros-Louis, F., et al. Hum MolGenet 17, 2691-2702 (2008)) was performed prior to first dosing and atweekly interval during the study. Testing was performed at 4-rpm speedwith 0.25 rpm/s acceleration. Each mouse was subjected to three trialsper session. The number of seconds each mouse remained on the rotarodapparatus for each trial was recorded.

The results are shown in FIGS. 4A and 4B. FIG. 4A shows the best scores(of the three trials) for individual mice in each group. FIG. 4B shows aregression analysis of the data in FIG. 4A. The data show that therotarod performance of the vehicle-treated group declined over the 15week period. However, the performance of both the 4-O-Methyl WA- and27-O-Methyl WA groups actually improved over that time period. Thelinear regression analysis showed statistical significance for both4-O-Methyl WA (p value of 0.02) and 27-O-Methyl WA (p value of 0.02),but not for the vehicle (p value of 0.2).

The results also demonstrate that the mice were able to tolerate theadministration of 5 mg/kg of the WA analogs every other day for 15weeks, providing further evidence for the safety of the compounds.

Example 7 Larger-Scale Synthesis and Purification of 27-O-MethylWithaferin A

27-O-methyl withaferin was prepared from withaferin A starting materialand purified using the procedures outlined below.

1. Preparation of 4,27-bis-O-triethylsilylwithaferin A

To a stirred solution of withaferin A (4.0 g) in N,N-dimethylformamide(40 mL) at 0° C. was added imidazole (2.31 g 4 eq.).Triethylchlorosilane (4.28 mL, 3 eq.) was added in small portions overapproximately 5 minutes, and the resulting mixture was stirred undernitrogen at 0° C. for approximately 2 hrs. After the reaction wascomplete as determined by HPLC, methanol (2 mL) was added and themixture was stirred for another 5-10 min. The reaction mixture wasdiluted with ethyl acetate (250 mL) and washed with brine (4×100 mL).The aqueous washings were combined and back extracted with ethyl acetate(2×100 mL). All ethyl acetate extracts were combined, washed again withbrine (3×100 mL), dried with anhydrous sodium sulfate and concentratedto obtain 10 g of crude 4,27-bis-O-triethylsilylwithaferin A as a paleoil.

2. Preparation of 4-O-triethylsilylwithaferin A

Crude 4,27-bis-O-triethylsilylwithaferin A (10 g) was dissolved inaqueous tetrahydrofuran (120 mL, THF/water, 9/1) at room temperature.Pyridinium p-toluenesulfonate (PPTS, 120 mg) was added. The mixture wasstirred for approximately 13 hours, during which additional PPTS(approximately 145 mg) was added in 6-30 mg portions to the reaction in1 hour intervals. The reaction progress was closely monitored by HPLC.After the reaction was complete, the mixture was diluted with ethylacetate (300 mL) and washed with brine (5×100 mL). The aqueous washingswere combined and back extracted with ethyl acetate (2×50 mL). All ethylacetate extracts were combined, washed with brine (100 mL), dried withanhydrous sodium sulfate, and concentrated to yield a crude product(11.5 g) as a pale gum. The crude product was purified by silica gelcolumn chromatography (dichloromethane/acetone 95-93/5-7) to afford 4.2g of pure 4-O-triethylsilylwithaferin A.

3. Preparation of 27-O-methyl-4-O-triethylsilylwithaferin A

4-O-triethylsilylwithaferin A (1.0 g) was dissolved in a mixture ofanhydrous THF (40 mL) and methyl iodide (8 mL) and cooled in anice-water bath under nitrogen. Sodium hydride (60% in mineral oil, 108mg, 1.58 eq.) was added and the mixture was stirred at room temperaturefor 4 minutes. The mixture was then stirred at room temperature forapproximately 1 hour 40 minutes, during which the reaction was closelymonitored by HPLC. After the product had reached 20-30%, the mixture wasdiluted with ethyl acetate (200 mL) and washed with brine (3×80 mL). Allwashings were combined and back extracted with ethyl acetate (2×50 mL).All ethyl acetate extracts were combined, washed with 10% aqueous sodiumthiosulfate (50 mL), brine (2×80 mL), dried over anhydrous magnesiumsulfate, and concentrated.

The crude product, pooled with another batch started with 0.5 g4-O-triethylsilylwithaferin A, was purified by column(dichloromethane/acetone 95-93/5-7) to give 413 mg of27-O-methyl-4-O-triethylsilylwithaferin A and 756 mg of recoveredstarting material. The average yield was about 27%.

4. Preparation of 27-O-methylwithaferin A

27-O-methyl-4-O-triethylsilylwithaferin A (413 mg) was dissolved in amixture of THF (9.5 mL) and pyridine (1.2 mL) and stirred in anice-water bath. Pyridine hydrofluoride (0.85 mL) was added dropwise, andafter 5 minutes at 0° C., the mixture was stirred at room temperaturefor 1.5 hr. The mixture was diluted with ethyl acetate (150 mL), washedwith 0.1 N hydrochloric acid (50 mL) and brine (2×50 mL). The washingswere combined and back extracted with ethyl acetate (2×50 mL). All ethylacetate extracts were combined and washed with saturated sodiumbicarbonate (50 mL), brine (2×50 mL), dried over sodium sulfate, andconcentrated. The crude product was purified by silicagel columnchromatography (hexane/acetone, 70-65/30-35) to yield 275 mg of27-O-methylwithaferin A (>95% purity; 82% yield). The purified productwas further recrystallized in acetone and hexane to increase the purityto approximately 99% as assessed by HPLC.

Example 8 Larger-Scale Synthesis and Purification of4-O-Methylwithaferin A

4-O-methylwithaferin A was prepared from withaferin A starting materialand purified using the following procedures.

1. Preparation of 27-O-(tert-butyldimethylsilyl)withaferin A

Withaferin A 5.6 g were dissolved in 60 mL of dichloromethane.Triethylamine (4.0 mL) was added to the mixing solution followed by theaddition of 4.01 g of tert-butyldimethylchlorosilane. The solution wasstirred at ambient temperature for approximately 80 hrs. The solutionwas washed with water and concentrated to give 6.95 g of crude27-O-(tert-butyldimethylsilylwithaferin A. This was crystallized frommethanol and dried to give 5.6 g of27-O-(tert-butyl.dimethylsilywithaferin A with an HPLC purity of 99.5%.Shorter reaction times can be achieved by the addition of 0.5 g ofdimethylaminopyridine leading to 95% completion in 12 hours.

2. Methylation of 27-O-(tert-butyldimethylsilyl)withaferin A

4.0 g of 27-O-(tert-butyldimethylsilyl)withaferin A was placed in around bottom flask under nitrogen. 20 mL of anhydrousN,N-dimethylformamide were added to the flask. Dissolution of the solidswas incomplete, but addition of 10 mL of methyl iodide yielded a clearsolution. Sodium hydride (0.32 g, 60% in mineral oil) was added into themixing solution over 10 minutes. HPLC showed approximately 80%completion. An additional 0.5 g of sodium hydride was added to achieve99% completion. Acetic acid was used to quench remaining sodium hydride.The mixture was diluted with dichloromethane and washed with water. Theorganic layer was concentrated to 20 mL. HPLC showed 88% purity of4-O-methyl-27-O-(tert-butyldimethylsilyl)withaferin A.

3. Preparation of 4-O-methylwithaferin A

The solution of 4-O-methyl-27-O-(tert-butyldimethylsilyl)withaferin Aobtained above was transferred to a PTFE flask rinsing forward with 30mL of dichloromethane. Pyridine (3 mL) was added, followed by 1 mL ofpyridine hydrofluoride. The reaction was monitored by HPLC. After 5.5hours, an additional 0.5 mL of pyridine hydrofluoride was added. Thereaction continued for another 3 hours to 92% completion. The reactionwas worked up by washing the organic solution with water, then sodiumbicarbonate solution, and finally sodium chloride solution. Thedichloromethane layer was concentrated on a rotary evaporator to give aviscous liquid. This was diluted with 36.5 mL of dichloromethane and36.5 mL methanol and washed with 73 mL water. The organic layer wasconcentrated under vacuum to give 3.9 g of crude 4-O-methylwithaferin A.

4. Purification of the 4-O-methylwithaferin A

The crude 4-O-methylwithaferin A obtained above was dissolved indichloromethane and loaded onto a 100 g silicagel column packed withdichloromethane. The column was eluted with 30% v/v acetone indichloromethane. The fractions were analyzed by HPLC and thosecontaining pure product were combined and evaporated to give 1.8 g ofsolids at 97% purity. The solids were crystallized from a mixture of 5mL methanol and 10 mL methy-tert-butylether. The solids were dried in avacuum oven to give 0.61 g of 4-O-methylwithaferin A at 98% HPLC purity.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A compound having a structure represented by Formula (Ia):

wherein: R¹ is hydrogen, or methyl; and R³ is hydrogen, or methyl, wherein, R¹ and R³ cannot both be hydrogen.
 2. The compound of claim 1 wherein R¹ is methyl and R³ is hydrogen.
 3. The compound of claim 1 wherein R¹ is hydrogen and R³ is methyl.
 4. The compound of claim 1 wherein R¹ is methyl and R³ is methyl.
 5. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient. 